INTRODUCTION:

Black medick(Medicago lupulina L.) belongs to the Medickgenus (Medicago L.) and to the Legume family (Fabaceae Lindl.). The legume family is a flower plant group of the most important human usefulness after grasses. The Medicago genre is a high protein, amino acid, vitamin, and trace elements folder plant widely used in the animal husbandry.¹ The M. lupulinaherb is traditionally used in folk medicine as an antiinflammatory, antiulcerogenic, antiseptic, cholagogic, diuretic, capillary fixating, antispasmodic, and antitumor drug.^2-5

In recent decades, triterpene glycosides and flavonoids are discovered in the M. lupulina composition.^6-8 Flavonoid composition of the M. lupulina herbal raw material includes: rutin, quercetin, luteolin, and luteolin-7-glycoside.⁹ Plant flavonoids inhibit all carcinogenesis stages and optimize action of antitumor medicines.¹⁰ Quercetin inhibits metabolic activation of suspected carcinogens and stimulates two stages of carcinogen metabolism.¹⁰ According to recent clinical research of summarized flavonoids and quercetin, introduction of the biologically active compounds reduces metastizing and invasiveness of cervical cancer.¹¹ This effect is probably caused by immunostimulant activity of the substance group.

It has been established that flavonoids of various chemical structure stimulate the NK-cells activity, so, they are immunomodulatory agents of a great potential. They can also be considered as perspective compounds for development of new medicines for treatment of tumour and viral diseases.¹²Currently, in clinical oncological practice, the recombinant lymphokines, especially interleukin-2 (IL-2), are used for the NK-cells activation. Interleukin-2 (IL-2) is also used as an independent drug as well as an ex-vivo lymphocyte activating drug for the following introducing (this is a so-called adoptive immunotherapy with the lymphokine-activated killers).¹³

In recent years, the lectins are found in the legumes. Lectins cause a statistically important cytotoxic action of mononuclear cells of peripheral blood on the Colo cells.¹⁴

Research objective:study of the effect of the M. lupulina extracton on cytotoxic antitumor activity of healthy donors and oncological patients lymphocytes.

MATERIALS AND METHODS:

M. lupulina herb was harvested at maximum active ingredient accumulation period (generative period) in the Podolsk district located in Moscow region of Russian Federation. The raw material ingredients were extracted in preparing liquid extract by accelerated and fractional maceration on the backflow principle 1:1. 70% ethyl alcohol was used as a fluid extraction. The extract was diluted ten times to concentration of 5 mg/ml, 1mg/ml, 0.1mg/ml, and 0.01mg/ml.

The M. lupulina herb extraction was standardized to quantificate a number of flavonoids in terms of rutin by spectrophotometric method in accordance with State Pharmacopoeia of the Russian Federation XIV edition (SPRF XIV ed.) based on the study of flavonoid composition of the M. lupulina herb and maximum UV absorption spectrum.

Flavonoids were divided and identified with a column LH-20 methanol balanced sephadex (sepharose-dekstan) chromatography with the following fraction chromatography made on the Silufol plates placed in the solvent system consisting of such solutions as acetic acid, water, and butanol (1:2:6). The detecting agent is 1% alcohol solution of aluminium chloride. 1-4 extracted fractions were studied by the TLC and the UV-spectroscopy methods. The TLC analysis (with such markers as standard samples of quercetin, rutin, and luteolin) of extract of the M. lupulina herb and 1-4 fractions extracted with sephadex chromatography showed that the third fraction contains rutin and the forth one contains quercetin mixed with rutin and unindentified substance similar to the luteolin-7-glycoside standard point which RF = 0.59 The UV- spectroscopy research of particular flavonoids and four fractions was with the Shimadzu UV- spectrophotometer and showed the flavonoid: rutin, quercetin, luteolin, and luteolin-7-glycoside spectrums. 210nm and 256nm ultraviolet absorption bands were general for all flavonoids. Rutin, luteolin, and luteolin-7-glycoside ultraviolet absorption bands were maximum at 372nm.⁹

Amount of flavonoids were analyzed with the spectrophotometer on rutin after the complexing reaction with aluminium chloride. Optical density of solution was measured on the spectrophotometer (Titertek Multiskan MCC/340 P Labsystem, Finland) at the relevant wave long. 95% alcohol aluminium chloride solution was used as a compared solution. Optical density of the State Standard Samples rutin complex was measured at the same time with that of aluminium chloride. Amount of the summarized flavonoids in terms of rutin and absolutely dried raw material was calculated as percent in accordance with the formula of SPRF XI ed., it was of 0.55±0.09% of the M. lupulina herbal extraction. Dried raw material weight loss was also determined by the method described in SPRF XI ed.; it was 9.55%.

The M. Lupulina extraction cytotoxic activity was studied with:

1) Mononuclear lymphocytes (ML) healthy donors and oncological patients

2) Line of K-562 human tumor cells and erythroleukemia (target-cells).

Mononuclear cells of twenty healthy donors and six oncological patients were extracted from peripheral anticoagulated (EDTA) venous human blood by the Boyum centrifugation method based on sedimentation in one stage density gradient of ficoll-urografin of 1.077 g/cm³ density.¹⁵After centrifugation with the horizontal centrifuge rotor of 400g, at room temperature, during 45 minutes, the mononuclear interphase nucleus was washed with medium 199 (Hanks' balanced salt solution) three times and centrifuged with the horizontal centrifuge rotor of 200g during 10minutes. Then, MLs were reconstituted in 1ml of the whole culture medium (RPMI 1640, 5% fetal calf serum, 40mcg/ml of gentamicin, 2mML of glutamine (PANECO) to concentration 10 x 10⁶kl/ml. Cells were accounted in the hemocytometer. The living cells were discovered with 0.1% trypan blue solution. Lymphocytes were extracted by incubating Ml in the culture plastic bottles (“COSTAR”) during 4 hours to remove the adherent macrophage cells.

Lines of K-562 human tumor cells were placed into 96-holed flat-bottom microplates (“COSTAR”) in number of 20000 cells by an hole into a 180µl culture medium hole consisting of RPMI-1640 with addition of 5% fetal calf serum, 40mcg/ml of gentamicin (“Pharmachim”), and 2mML of glutamine (Poliommelite and Viral Encephalitis Institute the RAS, Moscow).

The cytotoxic test (MTT assay) was used to determine the M. lupulina extraction cytotoxic activity of mononuclear lymphocytes of lines of K-562 human tumor cells. This is a sensitive colometric measurement method based on ability of mitochondrial dehydrogenases of living cells to convert water-soluble vital stain MTT 3-(4.5-dimethyl-2-thiazolyl) -2.5-diphenyl - 2H-tetrazolium bromide) (“Sigma”, МТТ, М2128)) into insoluble formazan crystallizing into the cells. Formazan concentration of solution measurement after attraction between formazan and dimethyl sulfoxide (DMSO) makes it possible to appreciate an amount of living cells and specific cell death induced by the damage agent in the cytotoxic study.¹⁶

A direct cytotoxic effect of extract on К-562 line cells was detected by introduction of various extract concentrations into cell culture: 5mg/ml, 1mg/ml, 0.1 mg/ml, 0.01mg/ml in amount of 20µl by the hole containing cell load in amount of 180µl by an hole (ethanol concentration in the holes was of 3.5%, 0.7%, and 0.07%). Every dilution was made with a triplet. There were three sets of tests during every experiment. In validation sets, 20µl of physiological solution or 20 µl of the physiological solution containing the ethanol concentrations appropriate for the tested ethanol concentration extracts was added to 180 ml of the whole culture medium. Optimal concentration of IL-2 (Roncoleukinum, BioTech, Russia) of 1000ea/ml was used as positive control.^13,16 After a 18-hourly CO₂incubation between target cells and extract at 37°С, 20µl of MTT solution (5mg/ml of pre-mixed phosphate buffered saline PBS kept at 4°С) was added to each hole. The plates covered with aluminium protection cover were placed into a dark place at 37°С temperature for MTT mitochondrial metabolism of K-562 living tumor cells. In for hours, the plates were centrifuged at 200g during five minutes to precipitate the cells of suspension culture of tumor cells, supernatant was removed carefully without touching cell sediment in two holes; 150µl of DMSO (“Sigma”) was added into each hole to solve the formazan crystals. A rotation shaker was used to solve formazan crystal more completely and quicker. Optical density of the solutions was measured with spectrophotometer (Titertec Multiscan MCC1340 Р, Flow Laboratory) at 540 nm.

Based on optical density of controlled and tested holes, a percent of dead target cells after incubation with the active substance, i.e. cytotoxic activity of tested samples was calculated by the following formula:

O.D.(kl.-target + ef.

% cytotoxicity = ------------------------------- 100 %

O.D.control kl. - target

Where

O.D. is optical density;

kl. – target is tumor К-562 line cells;

ef. – effector (lymphocyte)

The peripheral blood lymphocytes of six healthy donors and five oncological patients (suffered from lung cancer, breast cancer, and cervical cancer) were incubated with various extract concentrations and then they were added to the K-562 tumor line cells to identify Ml cytotoxic activity.

The study of activation influence of extract on secreting lymphocytes was pursued with lymphocytes of healthy donors. Lymphocytes were incubated at 37°С temperature during 48hours of extract concentration of 1 mg/ml and 0.1mg/ml. Then, the cytokine content in supernatant of the activated cells was detected.The test systems Quantik produced by the R&O company (Great Britain) were used during the study.

The statistical data was manipulated by variation statistics and determination of middle mark (M) of its error (m). A reliable difference between average values was valuated by the Student's t-test; the differences were reliable at р<0.05.

RESULTS AND DISCUSSION:

The extraction of M. lupulina has a direct cytotoxic effect on tumor cells at concentration of 5mg/ml; death of 41% of К-562 cells was observed, in comparison with 9% that of validation set at the effect of the whole culture medium including 3.5% of ethanol. At concentrations of 1mg/ml and 0.1mg/ml, cytotoxicity of cells was of 9-11%; the datadid not significantly differ from the validation set data (Table 1).

Table 1. The M. lupulina extraction cytotoxic effect on the K-562 tumor line cells

Set of tests	% cytotoxicity
Complete culture medium	8 ± 2
Complete culture medium +3.5% ethanol	9 ± 3
Complete culture medium +0.7% ethanol	3 ± 8
Complete culture medium +0.07% ethanol	7 ± 4
Extraction 1:2 (5mg/ml) 3.5% ethanol	41 ± 12*
Extraction 1:10 (1mg/ml) 0.7% ethanol	11 ± 6
Extraction 1:100 (0.1mg/ml) 0.07% ethanol	9 ± 4

* - Reliable changes at p<0.05 in comparison with the validation set, culture medium + 3.5% ethanol

Table 2 shows that incubation between all extraction dilutions and lymphocytes of healthy donors had a cytotoxic effect on the K-562 tumor line cells. A reliable increasing spontaneous lymphocyte cytotoxicity of healthy donors was on extraction dilution of 1.0mg/ml in comparison with the validation sets. Dilution of 0.1 mg/ml and 0.01mg/ml do not mainly influent on lymphocyte activity. An optimal dose of IL-2 was of more expressed stimulating action on spontaneous lymphocyte cytotoxic activity of healthy donors, than an extract concentration of 1.0mg/ml, however, statistics showed that the variations were unreliable.

Table 2. Modulating effect of the M. lupulina extraction cytotoxicity of healthy donors lymphocytes

Set of tests	% Cytotoxicity
Complete culture medium	37 ± 10
Complete culture medium + 3.5% ethanol	39 ± 3
Complete culture medium + 0.7% ethanol	33 ± 8
Complete culture medium + 0.07% ethanol	32 ± 4
Extract (1.0mg/ml) + 0.7% ethanol	51 ± 11*
Extract (0.1mg/ml) + 0.07% ethanol	40 ± 6
Extract (0.01mg/ml) + 0.007% ethanol	34 ± 2
IL-2 (1000ea/ml)	65 ± 9

* – Reliable changes at p<0.05 in comparison with the validation set, complete culture medium +0.7% ethanol

The similar course of antitumor cytotoxic activity is noted in condition of the lymphocyte oncological patients at the same extract dilution effect (Table 3). The original cytotoxicity of the lymphocytes was lower in comparison with that of healthy donors, however the extraction dilution of 1 mg/ml mostly increased killer properties of lymphocytes equitable with that of IL-2 effect.

Table 3. Modulating effect of the M. lupulina extraction on cytotoxicity of lymphocytes of oncological patients

Set of tests	% Cytotoxicity
Cell incubation medium	27 ± 12
Complete culture medium + 3.5% ethanol	29 ± 4
Complete culture medium + 0.7% ethanol	23 ± 9
Complete culture medium + 0.07% ethanol	24 ± 7
Extract (1.0 mg/ml) + 0.7 % ethanol	46 ± 11*
Extract (0.1 mg/ml) + 0.07 % ethanol	29 ± 3
Extract (0.01 mg/ml) + 0.007 % ethanol	28 ± 4
IL-2 (1000 ea/ml)	56 ± 9

* - Reliable changes at p < 0.05 in comparison with the validation set, complete culture medium + 0.7 % ethanol

The study has shown that 1.0mg/ml dilution tested extract sample increases of 12% in the level of interleukin-2 in healthy donors and of 17% in oncological patients in comparison with the validation set without effect of on the level of interleukin-1 (alpha and beta) reliably (Table 4).

CONCLUSION:

The extraction of M. lupulina has a direct cytotoxic effect on the line of K-562 human tumor cells in a concentration not lower than 5mg/ml; Increases the cytotoxicity of lymphocytes in healthy donors and oncological patients against K-562 tumor cells in a concentration of at least 1.0mg/ml. At this concentration, a reliable cytotoxic effect of lymphocytes of healthy donors increased at 18% in comparison with the validation set, that of oncological patients increased at 23%. The extraction of M. lupulina also activates the antitumor activity of natural killers by interleukin-2up-stimulation. The mechanism of M. lupulina activation influence on the cellular link of antitumor immunity can be related to the ability of biologically active substances isolated from alfalfa hops to stimulate release of regular cytokines from lymphocytes, inter alia, IL-2, related to the activation phenomenon of natural killers in lymphocyte population. Raw material of M. lupulina herb can be used to develop effective and low-toxic preventive medicines containing flavonoids.^17-26

CONFLICTS OF INTEREST:

None.

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Received on 06.08.2021 Modified on 21.10.2023

Research J. Pharm. and Tech 2024; 17(10):4658-4662.

DOI: 10.52711/0974-360X.2024.00717

Set of Tests	Concentration mg/ml
	IL-2		IL-1 alpha		IL-1 beta
	Healthy patients	Sick patients	Healthy patients	Sick patients	Healthy patients	Sick patients
1	2	3	4	5	6	7
Complete culture medium + 0.7% ethanol	9±2	12±4	6±2	16±7	2±0.9	12±9
Extract 1:10 (1.0 mg/ml) + 0.7 % ethanol	21±3*	29±3*	7±1	17±6	4±1	14±8
Extract 1:100 (0.1 mg/ml) + 0.07 % ethanol	10±3	17±7	3±2	18±9	2±1	16±8